Large Scale Chromatin Structure

大尺度染色质结构是指染色质在几百个碱基的尺度上的高阶折叠。人们对这种折叠知之甚少，但这种结构一定会影响DNA的转录、复制和修复。为了进一步了解这些结构，我们之前用光学显微镜检查了400 - 2000 kb的转录活性结构域。我们发现几个不同的结构域在光学显微镜图像中表现出相似的结构，即一系列相邻的点或直径约0.5微米的“珠子”。当这些相同的结构域转录不活跃时，只检测到一个点或“头”。因此，转录诱导从单珠状结构到多珠状结构的去浓缩。为了以更高的分辨率观察这些结构，我们正在探索x射线显微镜的适用性。这种成像方式可以在25纳米分辨率下显示整个未切片的哺乳动物细胞。染色质的检测依赖于有机物质相对于水的吸收所提供的自然对比。我们的目标是确定是否可以通过这种方法检测染色质结构，如果是这样，那么就可以提供25纳米分辨率的核染色质3D图像。我们目前的结果表明，至少通过透射x射线显微镜，没有足够的对比度来可视化染色质。然而，我们已经通过同样的方法获得了细胞质的非常详细的图像。我们现在可以通过分辨率为30纳米的x射线显微镜清楚地看到细胞中的所有膜结构。这种新方法的优点是，该方法实现了完整，未染色的3D标本的分辨率，从而提供了前所未有的细胞结构3D视图。我们目前正在考虑提高基于染色质结构的对比度的方法，包括开发相衬x射线显微镜或通过标准吸收x射线显微镜对冷冻核切片进行成像。

Large-scale chromatin structure is the higher-order folding of chromatin on the scale of several hundred kilobases. Little is known about this folding, but the structures must influence transcription, replication and repair of DNA. To learn more about these structures, we previously examined transcriptionally active domains of 400 - 2000 kb by light microscopy. We found that several different domains exhibit similar structures in light microscope images, namely a series of adjacent puncta or "beads" approximately 0.5 microns in diameter. When these same domains are transcriptionally inactive, only one punctum or "bead" is detected. Thus transcription induces a decondensation from single beaded to multi-beaded structures. To observe these structures at higher resolution we are exploring the applicability of x-ray microscopy. This imaging modality enables visualization of whole, unsectioned mammalian cells at 25 nm resolution. Detection of chromatin relies on the natural contrast afforded by the absorption of organic matter relative to water. Our goal is to determine if chromatin structure can be detected by this approach and if so, then to provide a 3D image of nuclear chromatin at 25 nm resolution. Our current results suggest that there is not sufficient contrast to visualize chromatin, at least by transmission x-ray microscopy. However, we have obtained strikingly detailed images of the cytoplasm by the same approach. We can now clearly visualize all membrane structures in the cell by x-ray microscopy down to 30 nm resolution. The advantage of this new approach is that the method achieves this resolution for intact, unstained 3D specimens, thereby providing an unprecedented 3D view of cellular architecture. We are presently considering ways to improve contrast of chromatin based structures, including development of phase contrast x-ray microscopy or imaging by standard absorption x-ray microscopy of frozen nuclear sections.